This is the first competitive renewal of GM067113, an investigation aimed at the long term goal of understanding the function and regulation of cullin-RING family of E3 ubiquitin ligases (CRLs). Looking back at the journey of the past several years, we are very pleased to see the contribution we made and more remarkably, the advancements achieved collectively by colleagues. Following the initial discovery of ROC1 and ROC2, we have made several findings toward this goal. These include: (1) that CAND1, an evolutionary conserved cullin-associated and neddylation dissociated protein, controls substrate recruitment to all CRLs, (2) that CUL3 binds to a BTB motif present in more than 200 mammalian proteins and potentially assembles a large number of BTB-CUL3-ROC1 ligases, (3) that DDB1-CUL4-ROC1 ligase target the replication licensing factor CDT1 for degradation in response to DNA damage, and (4) that CUL4 interacts with large number of cellular proteins, and (5) that pl70(KIAA080), an evolutionarily-conserved WD40 protein, binds abundantly with CUL4A via DDB1 and plays an essential role in cellular proliferation in a p53-dependent manner. With these series of findings as the basis, we propose a further research to elucidate the substrate- recruitment mechanism, the cellular function and regulation of CUL3- and CUL4-dependent ubiquitin ligases. The studies outlined in this proposal combine computer modeling, proteomic, biochemical, cellular and genetic approaches, offering an excellent and unique opportunity to help in the understanding of the largest family of E3 ubiquitin ligases. Three specific aims are: (i) to determine the functions and substrates of CRLS in mitosis and in transcriptional regulation, (ii) to determine the substrate recruiting mechanism of CRL4 ligases, and (iii) to determine the function and mechanism of p170.